In situ identification of keratin-hydrolyzing organisms in swine manure inoculated anaerobic digesters

Abstract

Feathers, a poultry byproduct, are composed of > 90% keratin which is resistant to degradation during anaerobic digestion. In this study, four 42-L anaerobic digesters inoculated with adapted swine manure were used to investigate feather digestion. Ground feathers were added into two anaerobic digesters for biogas production, whereas another two without feathers were used as negative control. Feather degradation and enhanced methane production were recorded. Keratin-hydrolyzing organisms (KHOs) were visualized in the feather bag fluids after boron-dipyrromethene (BODIPY) fluorescence casein staining. Their abundances correlated (R(2) = 0.96) to feather digestion rates. A 16S rRNA clone library was constructed for the bacterial populations attached to the feather particles. Ninety-three clones (> 1300 bp) were retrieved and 57 (61%) belonged to class Clostridia in the phylum Firmicutes, while 34 (37%) belonged to class Bacteroidia in the phylum Bacteroidetes. Four oligonucleotide FISH probes were designed for the major Clostridia clusters and used with other FISH probes to identify the KHOs. Probe FIMs1029 hybridized with most (> 80%) of the KHOs. Its targeted sequence perfectly matches that possessed by 10 Clostridia 16S rRNA gene clones belonging to a previously uncharacterized new genus closely related to Alkaliphilus in the subfamily Clostridiaceae 2 of family Clostridiaceae.

The fate of oxytetracycline in two‐phase and single‐phase anaerobic cattle manure digesters and its effects on microbial communities

“…Changes in the number of active microbial groups in digesters were characterized using fluorescent rRNA-targeted oligonucleotide probes specific to phylogenetically defined groups of selected bacteria (Gammaproteobacteria and Firmicutes) and methanogens (Methanobacteriales and Methanosarcinales), according to the most abundant groups of microorganisms detected in manure digesters. 14,15,29,37,38 The vitality of Gammaproteobacteria and Firmicutes were investigated in acidogenic digesters and compared with single-phase digesters. Similarly, the vitality of Methanobacteriales and Methanosarcinales were investigated in methanogenic digesters and compared with single-phase digesters.…”

In situ identification and quantification of protein-hydrolyzing ruminal bacteria associated with the digestion of barley and corn grain

“…BODIPY FL DQ TM casein staining was performed as previously described for biodigester sludge by Xia et al (2011) with slight modifications. Briefly, 400 µL of filtrated bag fluids were transferred into 1.5-mL Eppendorf tubes and centrifuged (4500 g for 10 min).…”

“…1A). BODIPY FL DQ TM casein staining combined with DAPI (4′,6-diamidino-2-phenylindole; Xia et al 2011) showed that these PHOs were more abundant (5-10% of total microbial cells) in the low concentrate than in the high concentrate diet (3-6% of total microbial cells), suggesting that they played a more important role in proteolysis of the low concentrate diet. We used BODIPY FL DQ TM casein staining combined with FISH (Xia et al 2007) to identify PHOs with CSA serine-and metallo-protease activity.…”

Microbial Insight into a Pilot-Scale Enhanced Two-Stage High-Solid Anaerobic Digestion System Treating Waste Activated Sludge

“…Clone SPAD53 was retrieved from a keratin (recalcitrant organic material) digestion system. In the system of Xia [ 51 ], a TAN concentration of 5.5–7.5 g/L was beneficial to the SAO pathway. However, it was difficult to precisely identify the family and genus categories of both Bac-OTU-17 and Bac-OTU-21 due to the lack of cultured relatives.…”